Abstract/Project Summary Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human herpesvirus, which causes Kaposi sarcoma (KS) as well as B cell lymphoproliferative disorders in the absence of adequate immune control. KSHV- associated tumors are a significant cause of morbidity and mortality in transplant patients and individuals with HIV-disease. The oral cavity is an important site for KSHV biology because saliva is believed to be the primary mode of person-to-person transmission for the virus. The tonsil and other oral lymphoid tissues represent a logical anatomical site for early infection events because they are in contact with saliva and are rich in target cell types for KSHV infection including endothelial cells and B lymphocytes. Despite this, the biology of KSHV in the human tonsil remains poorly understood. We have successfully isolated a variety of primary cell lineages from human tonsil specimens and have developed robust in vitro infection models for tonsil-derived lymphocytes and non- lymphocyte cell lines. The current proposal will leverage our library of tonsil lymphocyte specimens and cell lines in order to explore the fundamental biology of KSHV transmission in this niche. Using ex vivo susceptibility of tonsil-derived B lymphocytes as a surrogate for overall susceptibility of each tonsil specimen to KSHV infection, we will use a variety of molecular and cell biology techniques to examine sample-intrinsic factors that may influence the ability of KSHV to infect a new human host. These factors include: (1) host immunological parameters (2) host gene expression parameters (3) viral co-infections and (4) bacterial co-infections/microbiome communities. We will also examine how KSHV manipulates host cytokine signaling during early infection in order to determine whether cytokines can be manipulated to limit the establishment of KSHV infection in tonsil lymphocytes. Finally, we will determine how KSHV spreads within and between tonsil-derived cell types and determine which cell types are (1) important for transferring infection to disease-relevant cell types (2) potential sources of KSHV shedding into saliva. The results of this research will provide critical information about factors influencing KSHV transmission, and will highlight novel therapeutic targets that can be exploited to limit the spread of KSHV within the human population.